The goal of this R21 is to use our newly developed dual-color Fluorescence-tracing Orthotopic AlloTransplantation (FLOAT) system to investigate the molecular mechanisms underlying salivary gland cancer (SGC) progression. Bioinformatics studies of human SGC have identified putative molecular pathways that regulate tumor invasion, but follow-up analysis has proved challenging. Cell culture models are limited by the lack of a native tumor microenvironment and transgenic animal models are limited by resource-intensive methods and difficulties distinguishing tumor cells from the surrounding stroma. Therefore, new approaches that allow researchers to rapidly test molecular hypotheses, while preserving the native 3D structure of the tumor microenvironment, are needed. Furthermore, these models need to consider the impact of other comorbidities in distant sites, such as obesity. Our proposal will address this need using the FLOAT system, a simple and experimentally tractable orthotopic transplantation model, which allows researchers to visualize implanted salivary tumor cells and the adjacent microenvironment, to accurately model many aspects of in vivo SGC progression. This system utilizes DsRed fluorescence to label salivary tumor cells, which are transplanted into the salivary glands of green fluorescent protein-expressing mice. Dual-color fluorescence allows researchers to rapidly evaluate the tumor cells relative to the adjacent stroma. Our preliminary data demonstrate that the FLOAT system was useful for demonstrating that intrinsic tumor autophagy capacity modulates the architecture of tumor-bearing glands and survival. We will now use the FLOAT system to evaluate how obesity promotes local invasion of primary SGC lesions in vivo. Local invasion is required for SGC progression, but this multifaceted process is not well understood. Also, although changes in both tumor cells and the surrounding matrix have been reported during cancer progression, a direct mechanistic link between diet-induced obesity and accelerated SGC progression has not been established. We hypothesize that diet-induced obesity exacerbates SGC local invasion by inducing changes in the tumor microenvironment that permit or enhance local invasion. Given the crucial role of the tumor microenvironment in cancer progression, we further predict that the effect of diet-induced obesity on the type and activity of cells in the tumor microenvironment facilitates SGC invasion. In Aim 1, we will use the FLOAT system to test the effects of diet-induced obesity on SGC local invasion, gene expression, and lipid profiles. This Aim includes 3D matrix-assisted laser desorption/ionization (MALDI)-tissue-imaging mass spectrometry (TIMS) analysis of lipid profile heterogeneity in the invading tumor. In Aim 2, we will use the FLOAT system to test the effects of diet-induced obesity on fibrosis and inflammation in the microenvironment. The FLOAT system represents a powerful first step for both visualizing and distinguishing the heterogeneous components of host-tumor interaction during local invasion and a new system for efficiently testing novel therapeutics.